The overall objective of my laboratory is to elucidate pathways involved in the post-transcriptional regulation of gene expression. We have recently identified a 50kd protein which binds viral pre- mRNAs in a sequence-specific fashion. We have identified binding sites for this protein upstream of the HIV poly(A) signal and downstream of the SV40 polyadenylation signal. This RNA-protein interaction clearly influences the efficiency of 3' end processing, as it can substitute for the downstream element of the SV40 late polyadenylation signal. In addition, the binding of the 50kd protein leads to a site-specific RNA cleavage event near the protein binding site. We will evaluate the functional significance of the 50kd protein with respect to SV40 and HIV mRNA metabolism. Specifically, the role of the protein in 3' end formation, RNA stability, and transcription termination will be evaluated. The relationship of the 50kd protein to another downstream element-specific factor, the hnRNP C proteins, will also be explored. Second, we will purify and characterize the site-specific nuclease activity associated with the 50 kd protein. In addition to relating this information to the function of the 50kd protein in mRNA metabolism, the characterization of this unique activity may be exploitable for future RNA studies such as the "restriction mapping" of spliced families of genes. Finally, we will molecularly clone the 50kd protein cDNA, raise specific antibodies, and biochemically characterize the 50kd protein- RNA interaction. These studies will not only provide important reagents for future work, but will also provide the foundation to construct plausible models for the mechanism of action of this mRNP. This study has pertinence to many health related topics, as post- transcriptional controls play a role in numerous cellular processes. In addition, mutations in the poly(A) signal have been shown to be directly associated with disease (ie thalassemia) in humans. Finally, increasing our knowledge of HIV regulatory elements will provide new avenues for therapeutic control of AIDS.